CN106916823A - The aptamer of vibrio parahemolyticus and its kit and method of application and detection vibrio parahemolyticus - Google Patents
The aptamer of vibrio parahemolyticus and its kit and method of application and detection vibrio parahemolyticus Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
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Abstract
The invention provides a kind of aptamer of vibrio parahemolyticus and its kit and method of application and detection vibrio parahemolyticus, it is related to the technical field of biology sensor.The present invention provide aptamer, can specific identification vibrio parahemolyticus, can be used in the detection of vibrio parahemolyticus；The kit that the present invention is provided, with sensitivity very high and specificity, simple to operate, detection is quick, and as a result naked eyes are visible, is adapted to all laboratories and is promoted, and is to be capable of achieving the real-time live analysis to vibrio parahemolyticus without professional training.
Description
Technical field
The present invention relates to biosensor technology field, a kind of aptamer more particularly, to vibrio parahemolyticus and
Its application and the kit and method of detection vibrio parahemolyticus.
Background technology
Vibrio parahemolyticus (Vibrio parahaemolyticus, abbreviation V.p.) is a kind of gram-negative of halophagia
Property bacterium, belongs to vibrionaceae vibrio bacterial, and this bacterium strong adaptability can be grown on 5~44 DEG C of temperature, pH4.8~11.0, salinity 1~8%
In the environment of scope.During vibrio parahemolyticus is distributed widely in inshore seawater and grows aquatic products therein, for example shrimp, crab,
Scallop etc..Conventional detection method has been difficult to meet the actual demand to vibrio parahemolyticus field quick detection at present.
Most commonly culture of microorganism, the physio-biochemical characteristics according to vibrio parahemolyticus are identified, typically needed
Want Zengjing Granule, be separately cultured, the step such as biochemical test, serological Identification.These methods and resultses good stabilities and it is recognized as
The national standard method of detection, but experimental implementation is complicated, time-consuming, and 3~5 days are needed from identification is sampled, and sensitivity and spy
The opposite sex is not high, it is difficult to for field assay.Instrumental method mainly utilizes gas chromatography, high performance liquid chromatography etc., mainly
The characteristic spectrum of the metabolite of chemical composition or generation according to vibrio parahemolyticus is being detected.Such methods and resultses reliability
And sensitivity is high, but cumbersome sample pretreatment process, accurate expensive instrument and veteran operating personnel are needed, it is unfavorable
Promoted in basic unit.Detected using molecular biology method, including the most frequently used polymerase chain reaction PCR, ring mediation
Isothermal duplication LAMP etc., is detected by the specific expression gene for expanding vibrio parahemolyticus.This method is substantially reduced
Detection time and simplify detection program, but early stage needs to extract bacteria total DNA or RNA, and there is false positive higher
Rate.Immunological method is a kind of based on the principle specifically bound between antigen and antibody realization detection, and conventional having is enzyme-linked
Immunoabsorption ELISA, enzyme-linked XRF ELFA, electrochemical methods etc..Immunological detection method have high specificity,
Sensitivity is high, the advantages of be easy to observation, but prepare that the cycle of antibody is long, high cost, and is not suitable for preservation steady in a long-term, thus limit
Their extensive use is made.
Aptamer (Aptamer) is one section of oligonucleotides, by external artificial evolution's program
(Systematicevolution of ligands by exponential enrichment, SELEX) screening is obtained, and it can
With efficiently, specifically combine various parts, such as albumen, micromolecular compound, cell, specificity is as synantibody.Due to
It is easily-synthesized, the advantages of easily store and easily modify, have in medical diagnosis treatment, SARS drug design and analysis detection good
Good application prospect.But, how aptamer is preferably applied among the detection of vibrio parahemolyticus, it is necessary to enter one
The exploration and research of step.
In view of this, it is special to propose the present invention.
The content of the invention
First purpose of the invention is the aptamer for providing vibrio parahemolyticus, second object of the present invention
It is the application of the aptamer that vibrio parahemolyticus is provided, third object of the present invention is to provide secondary molten for detecting
The kit of courageous and upright vibrios, fourth object of the present invention is the method for providing detection vibrio parahemolyticus, existing to alleviate
Vibrio parahemolyticus detection sensitivity present in technology is low, unhandy technical problem.
The invention provides a kind of aptamer of vibrio parahemolyticus, including：The aptamer has such as SEQ
Sequence shown in ID NO.1：
GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA(SEQ ID NO.1)。
In addition, the application present invention also offers described aptamer in following I or II：
I, detection vibrio parahemolyticus；
II, prepare kit for detecting vibrio parahemolyticus.
In addition, present invention also offers a kind of kit for detecting vibrio parahemolyticus, the kit includes power
Profit requires aptamer and the not fully complementary cDNA of the aptamer described in 1, can be self-assembled into the two of long-chain
Individual hair fastener sequence H1 and H2 and protoferriheme；
The cDNA can trigger the self assembly of the H1 and H2；
3 ' the ends and 5 ' ends of the H1 and H2 have respectively can form the moiety precursor nucleotides sequence of G- tetramer structures
Row.
Further, the base number cDNA not complementary with the aptamer is 3-6.
Further, the cDNA with the sequence as shown in SEQ ID NO.2：
TAGACGCCACCCACACC(SEQ ID NO.2)。
Further, the 5 ' ends of the cDNA and H1 are complementary；3 ' the ends of the H1 are complementary with the 5 ' ends of the H2, institute
3 ' the ends for stating H2 are complementary with the 5 ' ends of the H1.
Further,
The H1 has the sequence as shown in SEQ ID NO.3：
AGGGCGGGGGTGTGGGGGCGTCTAACCCACACCTTCTTGTTGGGT(SEQ ID NO.3)；
The H2 has the sequence as shown in SEQ ID NO.4：
TGGGTAGACGCCACCCACACCAAGAAGACGGTGTGGGTGGGTAGGGCGGG(SEQ ID NO.4)；
After the H1 and H2 are self-assembled into long-chain, positioned at the moiety precursor nucleotide sequence at the H1 3 ' ends, and positioned at institute
The moiety precursor nucleotide sequence at the ends of H2 5 ' is stated, complete G- tetramer structures can be formed；Positioned at the portion at the H2 3 ' ends
Divide precursor nucleotides acid sequence, and positioned at the moiety precursor nucleotide sequence at the H1 5 ' ends, the complete G- tetramers can be formed
Structure；
After the complete G- tetramer structures combine the protoferriheme, can be formed with catalysis activity
DNAzyme。
Further, the kit also includes the substrate that can be catalyzed by the DNAzyme.
Further, the substrate is tetramethyl benzidine.
In addition, present invention also offers a kind of method of detection vibrio parahemolyticus is detected, methods described includes：
When in the absence of vibrio parahemolyticus, aptamer is not fully complementary with cDNA, and the cDNA is closed, and H1 is
Hairpin structure, H2 is hairpin structure；
In the presence of having vibrio parahemolyticus, vibrio parahemolyticus is combined with the aptamer, described
CDNA is exposed, and 5 ' the end complementary pairings of the cDNA the being exposed and H1, the hairpin structure of the H1 is opened, 3 ' ends
Exposure；3 ' the ends of the H1 hold complementary pairing, the hairpin structure of the H2 to be opened with the 5 ' of the H2,3 ' end exposures；It is described
3 ' the ends of H2 hold complementary pairing, the hairpin structure of the H1 to be opened with the 5 ' of the H1,3 ' end exposures；Self assembly successively is grown up
Chain；
After complementary pairings are held in the 3 ' ends of the H1 with the 5 ' of the H2, the G- tetra- that can be formed positioned at the H1 3 ' ends gathers
The moiety precursor nucleotide sequence of body structure, and positioned at the moiety precursor that can form G- tetramer structures at the H2 5 ' ends
Nucleotide sequence, can form complete G- tetramer structures；G- tetramer structures can be formed positioned at the H2 3 ' ends
Moiety precursor nucleotide sequence, and positioned at the moiety precursor nucleotides sequence that can form G- tetramer structures at the H1 5 ' ends
Row, can form complete G- tetramer structures；
After the complete G- tetramer structure combination protoferrihemes, the DNAzyme with catalysis activity can be formed,
The DNAzyme is capable of the change of catalytic substrate color, to realize the detection to the vibrio parahemolyticus；
The aptamer has the sequence as shown in SEQ ID NO.1：
GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA(SEQ ID NO.1)；
The cDNA has the sequence as shown in SEQ ID NO.2：
TAGACGCCACCCACACC(SEQ ID NO.2)；
The H1 has the sequence as shown in SEQ ID NO.3：
AGGGCGGGGGTGTGGGGGCGTCTAACCCACACCTTCTTGTTGGGT(SEQ ID NO.3)；
The H2 has the sequence as shown in SEQ ID NO.4：
TGGGTAGACGCCACCCACACCAAGAAGACGGTGTGGGTGGGTAGGGCGGG(SEQ ID NO.4)。
The present invention provide aptamer, can specific identification vibrio parahemolyticus, can be used in parahemolyticas
The detection of vibrios；The kit that the present invention is provided, with sensitivity very high and specificity, simple to operate, detection is quick, as a result
It is visually visible, it is adapted to all laboratories and is promoted, it is to be capable of achieving the real-time live to vibrio parahemolyticus without professional training
Analysis.
Brief description of the drawings
In order to illustrate more clearly of the specific embodiment of the invention or technical scheme of the prior art, below will be to specific
The accompanying drawing to be used needed for implementation method or description of the prior art is briefly described, it should be apparent that, in describing below
Accompanying drawing is some embodiments of the present invention, for those of ordinary skill in the art, before creative work is not paid
Put, other accompanying drawings can also be obtained according to these accompanying drawings.
Fig. 1 is each sequence and its structure of the kit that embodiment 1 is provided；
Fig. 2 is the schematic diagram of the detection method that embodiment 2 is provided；
Fig. 3 is the result figure of test experience in embodiment 3；
Fig. 4 is the result figure of test experience in embodiment 3.
Specific embodiment
Technical scheme is clearly and completely described below in conjunction with embodiment, it is clear that described reality
It is a part of embodiment of the invention to apply example, rather than whole embodiments.Based on the embodiment in the present invention, the common skill in this area
The every other embodiment that art personnel are obtained under the premise of creative work is not made, belongs to the model of present invention protection
Enclose.
Unless stated otherwise, " aptamer " of the present invention is " aptamer ".
Aptamer Aptamer is used as molecular recognition elements for the present invention, after capture object bacteria, by the DNA for building
Self assembly non-enzymatic amplification of signal system, for the hypersensitive Visual retrieval to vibrio parahemolyticus.The scheme that the present invention is provided
Design principle and thinking it is specific as follows：
(1) screening of specific recognition vibrio parahemolyticus Apatmer
Object bacteria enrichment and amplification of signal all rely on the affinity and specificity of selected aptamer in this experiment.It is logical
It is positive sieve bacterium to cross using vibrio parahemolyticus, and salmonella, Listeria and Escherichia coli carry out Cell-SELEX for negative sieve bacterium
Screening, is that the detection of next step is established so as to filter out the specific nucleic acid aptamers of energy specific recognition vibrio parahemolyticus
Basis.The dissociation constant Kd values of the aptamer and purpose thalline for screening are calculated by flow cytometry analysis, from
The middle selection most strong nucleotide sequence of two adhesions, the then Aptamer for needed for.The Aptamer sequences of acquisition are used in next step
Hold the strand replacement reaction of (2).
(2) the Aptamer strand replacement reactions of vibrio parahemolyticus mediation
It is identification molecule, the design DNA sequence dna complementary with Aptamer, the secondary haemolysis of analysis with vibrio parahemolyticus Aptamer
Property vibrios mediation strand replacement reaction.Closing on end modified corresponding fluorophor and being quenched in Aptamer and complementary DNA
Group, in the presence of object bacteria, can replace Aptamer from complementary double-strand, be evaluated by analysis of fluorescence intensity
Strand displacement effect.After vibrio parahemolyticus and Aptamer specificity stable bonds, the complementary DNA being closed then is exposed,
So as to start the DNA self assembly signal amplification process of next step content (3).
(3) structure of the universal amplification of signal system of DNA self assemblies
Based on the DNA self assemblies of stem ring hairpin structure (Hairpin), a kind of universal amplification of signal system is designed.This
Part is needed to analyze the structure of Hairpin, and evaluating Hairpin by fluorescence method is combined startup self assembling process with cDNA, is evaluated
Effects of the cDNA in cyclic amplification.Research starts influence of the length of chain to self-assembling reaction, can observe face by colorimetric method
Color change power is analyzed self assembly efficiency.It is changed by the stem ring length to Hairpin, sequence, carrys out comparison signal
The change of response in the front and rear colorimetric method of amplification, to optimize the self assembly efficiency of Hairpin.The analysis Hairpin self assembly times,
The influence of reaction system buffer solution and reaction temperature to amplification system.Such self assembly can be by the parahemolyticas arc of trace
Bacterium signal efficiently amplifies, and a large amount of Self-assembled DNA products of generation will be used to start the visual analyzing of next step content (4).
(4) design of visual analyzing platform and realization
Using the peroxidase activity feature of G-quadruplex, using G-quadruplex as the biography of visual analyzing
Sensing unit, by Haripin stem of the G- tetramer sequences design in content (3), enables the single stranded DNA produced in content (2)
Open the loop-stem structure of Hairpin and activate the G- tetramers.The G- tetramers after activation have DNAzyme catalysis activities, by nothing
The soluble product of color substrate oxidation au bleu, directly observes by the naked eye experimental result, is suitable to field quick detection analysis.It is right
The actual sample bought on the market is analyzed, and by testing result and traditional biochemical culture method, instrument analytical method etc.
It is compared, verifies the accuracy of the nucleic acid sensing platform, carries out error analysis, the main ginseng in one-step optimization of going forward side by side experiment
Number, stabilization of nucleic acids sensing.
Compared with prior art, the scheme of the detection vibrio parahemolyticus that the present invention is provided has following advantage：
(1) polymerase need not be added in system of the present invention carries out amplification of signal, reduces background signal.
(2) without carrying out any modification to target sequence in system of the present invention, and when vibrio parahemolyticus is mixed in reality
Still signal can be detected in aquatic products sample, the method can be used for the quantitative determination of actual sample.
(3) double-strand being self-assembly of with DNA in the present invention amplifies mode as signal, reacts quick and convenient, shortens
Operating time.
(4) the non-enzyme assay method of DNA self assemblies of the present invention has sensitivity very high, and detection is limited to 0.1fM, than
The method reported is higher by more than 10 times, range of linearity 0.1fM-1nM.Detection is rapid convenient, and direct Macroscopic analysis have saved detection
Cost.
(5) aptamer identification target bacterium of the present invention has specificity very high, and other materials are to detecting not
Produce influence.
(6) by changing nucleotide sequence, the described method of invention can be used for a series of different food borne pathogenses of detection
Microorganism.
Embodiment 1 is used to detect the kit of vibrio parahemolyticus
A kind of kit for detecting vibrio parahemolyticus is present embodiments provided, kit includes following material：
Aptamer, with the sequence as shown in SEQ ID NO.1：
5’-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3’(SEQ ID NO.1)；
CDNA, with the sequence as shown in SEQ ID NO.2：
5’-TAGACGCCACCCACACC-3’(SEQ ID NO.2)；
H1, with the sequence as shown in SEQ ID NO.3：
5’-AGGGCGGGGGTGTGGGGGCGTCTAACCCACACCTTCTTGTTGGGT-3’(SEQ ID NO.3)；
H2, with the sequence as shown in SEQ ID NO.4：
5’-TGGGTAGACGCCACCCACACCAAGAAGACGGTGTGGGTGGGTAGGGCGGG-3’(SEQ ID
NO.4)；
Protoferriheme；
Tetramethyl benzidine (TMB).
Wherein, aptamer and the not fully complementary combinations of cDNA, each self-forming hairpin structures of H1 and H2.
Fig. 1 show each sequence and structure.
The method that embodiment 2 detects vibrio parahemolyticus
The application method of the kit of the offer of embodiment 1 is be provided, is comprised the following steps：
(1) after the 95 DEG C of water-bath 5min of the vibrio parahemolyticus aptamer hair fasteners sequence containing identification for returning will be synthesized, slowly drop
Warm to room temperature, close hairpin structure.
(2) to the PBS dilution bacterium solutions that 2 μ L are added in system, room temperature reaction 30min so that vibrio parahemolyticus and hair fastener
In aptamer combine and open hairpin structure.
(3) hairpin structure opened adds H1 and H2 hair fasteners to mix by cDNA sequence separate out into single-chain state, in system
Conjunction liquid, room temperature reaction 1 hour, DNA carries out self-assembling reaction.
(4) to Hemin, room temperature reaction 10min is added in system, in allowing Hemin fully to mix self assembly double-strand.
(5) to adding Substrate cocktail A+B (wherein, A is for tetramethyl benzidine TMB, B are hydrogen peroxide), room temperature in system
Reaction 5-10 minutes, it is seen that color, when color is no longer deepened, adds H by colourless change au bleu2SO4Terminating reaction.Detection is former
Reason is as shown in Figure 2.
The detection of the target vibrio parahemolyticus of the various concentrations of embodiment 3
The present embodiment detected for the target vibrio parahemolyticus of various concentrations, specifically includes herein below：
The standard liquid of vibrio parahemolyticus is prepared, final concentration is respectively 100cfu, and 1000cfu, 10000cfu room temperature are protected
Deposit.
The bacterium solution of various concentrations is added separately in the reaction system in embodiment 2 described in (2), fully reaction, detection
Photon absorbing intensity and visual color change at 490nm.
Be can be seen that when there is vibrio parahemolyticus from the testing result of Fig. 3, color is by colourless change au bleu (in figure
It is not shown), light absorption value substantially increases；And with the increase of bacterial concentration, light absorption value increases.Wherein, Hemin blank groups are for only
Hemin adds magnetic bead, is not added with the DNA product of early stage self assembly, and magnetic bead blank group is only magnetic bead, is not added with the DNA of early stage self assembly
Product.
In order to assess the sensitivity of this detection method, we are configured with the vibrio parahemolyticus of various concentrations, from 10cfu
To 1015cfu, and determine the light absorption value of detection.The vibrio parahemolyticus as 10cfu is can be seen that from the testing result of Fig. 4
In the presence of, light absorption value is more than the three times of negative control reading, to illustrate test limit 10cfu of the method to vibrio parahemolyticus,
More than 10 times is higher by than traditional detection method.Also, as the increase of bacterial concentration is (from 10cfu to 1015Cfu), light absorption value
With increase, the logarithm (log of cell concentration10) with light absorption value be in good linear relationship, its range of linearity be from 10cfu to
109cfu。
Wherein, the abscissa of Fig. 4 represents the logarithm (log of vibrio parahemolyticus concentration10), in the ordinate of Fig. 3 and Fig. 4
" Abs " represents light absorption value.
For vibrio parahemolyticus pollution range it is wide, outburst rate is high, endanger big the characteristics of, present invention aptamer
Aptamer, by the DNA self assembly non-enzymatic amplification of signal systems for building after capture object bacteria, is used for as molecular recognition elements
To the hypersensitive Visual retrieval of vibrio parahemolyticus.The nucleic acid sensor can use at normal temperatures, it is simple to operation, detect into
This is relatively low, result naked eyes are visible, it is adaptable to the pollution condition of vibrio parahemolyticus in the quick analysis seafood in scene；The colorimetric method point
Analysis system is with a wide range of applications in food safety detection.And it is used for pair there is presently no report DNA self-assembling techniques
The detection of hemolytic vibrios, because the advantage of this technology is it is considered that it can be to meet modern vibrio parahemolyticus well
The requirement of Site Detection.
Finally it should be noted that：Various embodiments above is merely illustrative of the technical solution of the present invention, rather than its limitations；To the greatest extent
Pipe has been described in detail with reference to foregoing embodiments to the present invention, it will be understood by those within the art that：Its according to
The technical scheme described in foregoing embodiments can so be modified, or which part or all technical characteristic are entered
Row equivalent；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention technology
The scope of scheme.
SEQUENCE LISTING
<110>University Of Qingdao
<120>The aptamer of vibrio parahemolyticus and its kit and method of application and detection vibrio parahemolyticus
<130> 2017
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 36
<212> DNA
<213>Artificial sequence
<400> 1
gatcgggtgt gggtggcgta aagggagcat cggaca 36
<210> 2
<211> 17
<212> DNA
<213>Artificial sequence
<400> 2
tagacgccac ccacacc 17
<210> 3
<211> 45
<212> DNA
<213>Artificial sequence
<400> 3
agggcggggg tgtgggggcg tctaacccac accttcttgt tgggt 45
<210> 4
<211> 50
<212> DNA
<213>Artificial sequence
<400> 4
tgggtagacg ccacccacac caagaagacg gtgtgggtgg gtagggcggg 50
Claims (10)
1. a kind of aptamer of vibrio parahemolyticus, it is characterised in that including：The aptamer has such as SEQ ID
Sequence shown in NO.1：
GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA(SEQ ID NO.1)。
2. application of the aptamer described in claim 1 in following I or II：
I, detection vibrio parahemolyticus；
II, prepare kit for detecting vibrio parahemolyticus.
3. a kind of kit for detecting vibrio parahemolyticus, it is characterised in that the kit is included described in claim 1
Aptamer and the not fully complementary cDNA of the aptamer, two hair fastener sequences of long-chain can be self-assembled into
H1 and H2 and protoferriheme；
The cDNA can trigger the self assembly of the H1 and H2；
3 ' the ends and 5 ' ends of the H1 and H2 have respectively can form the moiety precursor nucleotide sequence of G- tetramer structures.
4. kit according to claim 3, it is characterised in that the alkali cDNA not complementary with the aptamer
Base number is 3-6.
5. kit according to claim 4, it is characterised in that the cDNA with as shown in SEQ ID NO.2
Sequence：
TAGACGCCACCCACACC(SEQ ID NO.2)。
6. kit according to claim 3, it is characterised in that the 5 ' ends of the cDNA and H1 are complementary；The H1
3 ' ends it is complementary with the 5 ' of H2 ends, the 3 ' ends of the H2 and the 5 ' ends of the H1 are complementary.
7. kit according to claim 6, it is characterised in that
The H1 has the sequence as shown in SEQ ID NO.3：
AGGGCGGGGGTGTGGGGGCGTCTAACCCACACCTTCTTGTTGGGT(SEQ ID NO.3)；
The H2 has the sequence as shown in SEQ ID NO.4：
TGGGTAGACGCCACCCACACCAAGAAGACGGTGTGGGTGGGTAGGGCGGG(SEQ ID NO.4)；
After the H1 and H2 are self-assembled into long-chain, positioned at the moiety precursor nucleotide sequence at the H1 3 ' ends, and positioned at the H2
The moiety precursor nucleotide sequence at 5 ' ends, can form complete G- tetramer structures；Positioned at the moiety precursor at the H2 3 ' ends
Nucleotide sequence, and positioned at the moiety precursor nucleotide sequence at the H1 5 ' ends, complete G- tetramer structures can be formed；
After the complete G- tetramer structures combine the protoferriheme, the DNAzyme with catalysis activity can be formed.
8. kit according to claim 7, it is characterised in that the kit also includes can be by the DNAzyme
The substrate of catalysis.
9. kit according to claim 8, it is characterised in that the substrate is tetramethyl benzidine.
10. a kind of method that detection detects vibrio parahemolyticus, it is characterised in that methods described includes：
When in the absence of vibrio parahemolyticus, aptamer is not fully complementary with cDNA, and the cDNA is closed, and H1 is hair fastener
Structure, H2 is hairpin structure；
In the presence of it there is vibrio parahemolyticus, vibrio parahemolyticus is combined with the aptamer, the cDNA
It is exposed, 5 ' the end complementary pairings of the cDNA the being exposed and H1, the hairpin structure of the H1 is opened, and 3 ' ends are sudden and violent
Dew；3 ' the ends of the H1 hold complementary pairing, the hairpin structure of the H2 to be opened with the 5 ' of the H2,3 ' end exposures；The H2
3 ' ends hold complementary pairings, the hairpin structure of the H1 to be opened with the 5 ' of the H1,3 ' end exposures；Self assembly successively is grown up
Chain；
After complementary pairings are held in the 3 ' ends of the H1 with the 5 ' of the H2, G- tetramer knots can be formed positioned at the H1 3 ' ends
The moiety precursor nucleotide sequence of structure, and positioned at the moiety precursor nucleosides that can form G- tetramer structures at the H2 5 ' ends
Acid sequence, can form complete G- tetramer structures；Positioned at the part that can form G- tetramer structures at the H2 3 ' ends
Precursor nucleotides acid sequence, and positioned at the moiety precursor nucleotide sequence that can form G- tetramer structures at the H1 5 ' ends, energy
Enough form complete G- tetramer structures；
After the complete G- tetramer structure combination protoferrihemes, the DNAzyme with catalysis activity can be formed, it is described
DNAzyme is capable of the change of catalytic substrate color, to realize the detection to the vibrio parahemolyticus；
The aptamer has the sequence as shown in SEQ ID NO.1：
GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA(SEQ ID NO.1)；
The cDNA has the sequence as shown in SEQ ID NO.2：
TAGACGCCACCCACACC(SEQ ID NO.2)；
The H1 has the sequence as shown in SEQ ID NO.3：
AGGGCGGGGGTGTGGGGGCGTCTAACCCACACCTTCTTGTTGGGT(SEQ ID NO.3)；
The H2 has the sequence as shown in SEQ ID NO.4：
TGGGTAGACGCCACCCACACCAAGAAGACGGTGTGGGTGGGTAGGGCGGG(SEQ ID NO.4)。
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| CN110093350A (en) * | 2019-01-17 | 2019-08-06 | 江南大学 | A kind of the optimization aptamers sequence and its application of specific recognition vibrio parahemolyticus |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110093350A (en) * | 2019-01-17 | 2019-08-06 | 江南大学 | A kind of the optimization aptamers sequence and its application of specific recognition vibrio parahemolyticus |
| CN110093350B (en) * | 2019-01-17 | 2022-10-18 | 江南大学 | Optimized aptamer sequence for specifically recognizing vibrio parahaemolyticus and application thereof |
| CN110286216A (en) * | 2019-05-16 | 2019-09-27 | 中国科学院武汉物理与数学研究所 | A kind of hemolytic relative gene O157:H7 detection method of quick visualization |
| CN111690648A (en) * | 2020-06-23 | 2020-09-22 | 福州市长乐区宝爱冬医学技术有限公司 | Sequence and application of nucleic acid aptamer TDHA for specifically recognizing vibrio parahemolyticus TDH |
| CN111690648B (en) * | 2020-06-23 | 2022-06-28 | 丽水君弘生物科技有限公司 | Sequence and application of nucleic acid aptamer TDHA for specifically recognizing vibrio parahaemolyticus TDH |
| CN111849995A (en) * | 2020-08-04 | 2020-10-30 | 福州金域医学检验所有限公司 | Aptamer TLH01 of thermolabile hemolysin TLH and application thereof |
| CN116515962A (en) * | 2023-06-26 | 2023-08-01 | 中国农业大学 | Self-extension isothermal amplification method of clover structure and application thereof |
| CN116515962B (en) * | 2023-06-26 | 2023-09-12 | 中国农业大学 | Self-extension isothermal amplification method of clover structure and application thereof |
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